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Analysis of Protein Posttranslational Modifications Using DIGE-Based Proteomics

  • Robert M. DeKroon
  • Jennifer B. Robinette
  • Cristina Osorio
  • Joseph S. Y. Jeong
  • Eric Hamlett
  • Mihaela Mocanu
  • Oscar AlzateEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 854)

Abstract

Difference gel electrophoresis (DIGE) is most often used to assess relative changes in the expression levels of individual proteins in multiple complex samples, and this information is valuable in making inferences about relative protein activity. However, a protein’s activity is not solely dependent upon its expression level. A change in activity may also be influenced by myriad posttranslational modifications (PTMs), including palmitoylation, ubiquitination, oxidation, and phosphorylation. In this chapter, we describe the use of DIGE to determine specific PTMs by introducing specific labels or changes in pI and/or molecular weight.

Key words

Difference gel electrophoresis Neuroproteomics Oxidation Palmitoylation Phosphory-lation Posttranslational modification Ubiquitination 

Notes

Acknowledgments

This work was supported by the UNC Systems-Proteomics Center and the Duke Neuroproteomics Center.

References

  1. 1.
    Yamagata A, Kristensen DB, Takeda Y, Miyamoto Y, Okada K, Inamatsu M, Yoshizato K (2002) Mapping of phosphorylated proteins on two-dimensional polyacrylamide gels using protein phosphatase. Proteomics 2: 1267–1276.PubMedCrossRefGoogle Scholar
  2. 2.
    Sun J, Morgan M, Shen R-F, Steenbergen C and Murphy E (2007) Preconditioning Results in S-Nitrosylation of Proteins Involved in Regulation of Mitochondrial Energetics and Calcium Transport. Circ Res 101: 1155–1163.PubMedCrossRefGoogle Scholar
  3. 3.
    Alban A, David SO, Bjorkesten L, Andersson C, Sloge E, Lewis S, and Currie I (2003) A novel experimental design for comparative two-dimensional gel analysis: two-dimensional difference gel electrophoresis incorporating a pooled internal standard. Proteomics 3: 36–44.PubMedCrossRefGoogle Scholar
  4. 4.
    Diez R, Herbstreith M, Osorio C, Alzate O (2009) 2-D Fluorescence Difference Gel Electrophoresis (DIGE) in neuroproteomics. In: Alzate O (ed) Neuroproteomics, CRC Press, Boca Raton.Google Scholar
  5. 5.
    DeKroon RM, Osorio C, Robinette JB, Mocanu M, Winnik WM, Alzate O (2011). Simultaneous detection of changes in protein expression and oxidative modification as a function of age and APOE genotype. J Proteome Res 10: 1632–1644.PubMedCrossRefGoogle Scholar
  6. 6.
    Storey JD and Tibshirani R (2003) Statistical significance for genomewide studies. PNAS 100: 9440–9445.PubMedCrossRefGoogle Scholar
  7. 7.
    Sanchez JC, Chiappe D, Converset V, Hoogland C, Binz PA, Paesano S, Appel RD, Wang S, Sennitt M, Nolan A, Cawthorne MA, Hochstrasser DF (2001) The mouse SWISS-2D PAGE database: a tool for proteomics study of diabetes and obesity. Proteomics 1: 136–163.PubMedCrossRefGoogle Scholar
  8. 8.
    Zhoa YM, Basu U, Dodson MV, Basarab JA, Guan LL (2010) Proteome difference associated with fat accumulation in bovine subcutaneous adipose tissues. Proteome Sci 8: 1–14.CrossRefGoogle Scholar
  9. 9.
    Babu GJ, Wheeler D, Alzate O, Periasamy M (2004) Solubilization of membrane proteins for two-dimensional gel electrophoresis: identification of sarcoplasmic reticulum membrane proteins. Anal Biochem 325: 121–125.PubMedCrossRefGoogle Scholar
  10. 10.
    Blackstone CD, Moss SJ, Martin LJ, Levey AI, Price DL, Huganir RL (1992) Biochemical characterization and localization of a non-N-methyl-D-aspartate glutamate receptor in rat brain. J Neurochem 58: 1118–1126.PubMedCrossRefGoogle Scholar
  11. 11.
    Lau LF, Mammen A, Ehlers MD, Kindler S, Chung WJ, Garner CC, Huganir RL (1996) Interaction of the N-methyl-D-aspartate receptor complex with a novel synapse-associated protein, SAP102. J Biol Chem 271: 21622–21628.PubMedCrossRefGoogle Scholar
  12. 12.
    Wessel D and Flugge UI (1983) A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Anal Biochem 138: 141–143.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Robert M. DeKroon
    • 1
  • Jennifer B. Robinette
    • 2
  • Cristina Osorio
    • 2
  • Joseph S. Y. Jeong
    • 1
  • Eric Hamlett
    • 1
  • Mihaela Mocanu
    • 2
  • Oscar Alzate
    • 1
    Email author
  1. 1.Department of Cell and Developmental BiologyUniversity of North CarolinaChapel HillUSA
  2. 2.UNC Systems-Proteomics Center, Program of Molecular Biology and Biotechnology, School of MedicineUniversity of North CarolinaChapel HillUSA

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